Blood clotting proceeds through a complex and dynamic biological pathway of interdependent biochemical reactions, referred to as the coagulation cascade, in which three pathways, the extrinsic pathway, the intrinsic pathway and the common pathway are included. Factor VIII (FVIII) is a glycoprotein, circulating as an inactive cofactor with full-length von Willebrand factor (vWF) in plasma in 1:50 ratio, that plays a critical role in the intrinsic pathway for maintaining normal hemostasis. Full-length FVIII is a non-covalently bound heterodimer comprised of a heavy chain (A1-A2-B domains) and light chain (A3-C1-C2 domains). In response to injury, the activated form of factor VIII separates from vWF and forms a complex with factor IXa and Factor X (so-called Xase complex) on the charged phospholipid membranes provided by activated platelets. The Xase complex further activates Factor V (FV) to generate FVa, which in turn activates prothrombin to thrombin together with Factor Xa and other components in the coagulation cascade to generate a stable clot.
Hemophilia A is a congenital X chromosome-linked bleeding disorder characterized by a deficiency in FVIII activity. Diminished FVIII activity inhibits the positive feedback loop in the coagulation cascade, ultimately leading to bleeding episodes with increased duration, extensive bruising, spontaneous oral and nasal bleeding, joint stiffness and chronic pain, and possibly internal bleeding and anemia, in severe cases. The most common treatment for Hemophilia A is replacement therapy with either human plasma-derived or recombinant FVIII through intravenous administration.
High production of recombinant FVIII (rFVIII) in mammalian cells has been found to be difficult. The large molecular weight of this protein (˜300 kDa), the complexity of the post-translational modifications required (e.g., numerous glycosylation and tyrosine sulfation sites), and the limits of expression elements (like mRNA instability) make the high production of rFVIII a challenge. Removal of the central B-domain has greatly improved rFVIII production, but the commercial production level of rFVIII is still generally in the range of 20-50 IU/ml. Additional possible factors contributing to the low expression level of the recombinant FVIII in mammalian cells may include, specifically or non-specifically, binding to expression cell membranes and FVIII instability (Kaufman, 1989, Mol. Cell. Biol., vol. 3, pp. 1233-1242: U.S. Pat. No. 8,759,293).
U.S. Pat. No. 8,759,293 discloses a method for preparing FVIII. FIG. 6 of the '293 Patent illustrates a first plasmid expression vector that expresses (i) mature vWF or truncated vWF domain-Fc fusion polypeptides, and (ii) propeptide sequences from independent promoters, and a second and a different plasmid expression vector the expresses (iii) human FVIII using a different selectable marker. The first and the second plasmids are co-transfected and taken up into mammalian cells under selection to create a stable cell line that expresses (i) vWF or vWF-Fc, (ii) vWF propeptide, and (iii) FVIII. The problem for this method is that each selected cell transfectant is obtained by independent and separate entry of FVIII and vWF-Fc plasmids into a mammalian cell. Selected cells can have different ratios of each plasmid. It is difficult to obtain cells reliably and reproducibly with consistent expression of proteins if the ratio of the transfected plasmids is unknown, and thus the absolute levels of the expressed proteins will change over time. For example, a primary cell transfectant produced by sequential transfection may have 10 copies of FVIII and 30 copies of vWF-Fc. Over-expression of vWF-Fc over FVIII, would result in excessive quantities of vWF-Fc relative to FVIII, and thus make downstream purification of FVIII difficult. Further, if the ratio of the two plasmids changes over time, a cell may become undesirable due to reduction in FVIII plasmids, while maintaining a constant number of vWF plasmids. The loss of one plasmid relative to another may upset the balance in expression levels, which results in difficult purification scenarios.
There is a need for improvement in the quality and quantity of recombinant FVIII available to patients. There is a need for improving the expression of FVIII and improving the yield of expressed FVIII after purification.